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. 2017 Apr 7;14(1):80.
doi: 10.1186/s12974-017-0859-9.

Exposure to gestational diabetes mellitus induces neuroinflammation, derangement of hippocampal neurons, and cognitive changes in rat offspring

Billy Vuong  1   2 , Gary Odero  1   2 , Stephanie Rozbacher  2 , Mackenzie Stevenson  2 , Stephanie M Kereliuk  1   3 , Troy J Pereira  1   3 , Vernon W Dolinsky  1   3 , Tiina M Kauppinen  4   5   6
Affiliations

Exposure to gestational diabetes mellitus induces neuroinflammation, derangement of hippocampal neurons, and cognitive changes in rat offspring

Billy Vuong et al. J Neuroinflammation. .

Abstract

Background: Birth cohort studies link gestational diabetes mellitus (GDM) with impaired cognitive performance in the offspring. However, the mechanisms involved are unknown. We tested the hypothesis that obesity-associated GDM induces chronic neuroinflammation and disturbs the development of neuronal circuitry resulting in impaired cognitive abilities in the offspring.

Methods: In rats, GDM was induced by feeding dams a diet high in sucrose and fatty acids. Brains of neonatal (E20) and young adult (15-week-old) offspring of GDM and lean dams were analyzed by immunohistochemistry, cytokine assay, and western blotting. Young adult offspring of GDM and lean dams went also through cognitive assessment. Cultured microglial responses to elevated glucose and/or fatty acids levels were analyzed.

Results: In rats, impaired recognition memory was observed in the offspring of GDM dams. GDM exposure combined with a postnatal high-fat and sucrose diet resulted in atypical inattentive behavior in the offspring. These cognitive changes correlated with reduced density and derangement of Cornu Ammonis 1 pyramidal neuronal layer, decreased hippocampal synaptic integrity, increased neuroinflammatory status, and reduced expression of CX3CR1, the microglial fractalkine receptor regulating microglial pro-inflammatory responses and synaptic pruning. Primary microglial cultures that were exposed to high concentrations of glucose and/or palmitate were transformed into an activated, amoeboid morphology with increased nitric oxide and superoxide production, and altered their cytokine release profile.

Conclusions: These findings demonstrate that GDM stimulates microglial activation and chronic inflammatory responses in the brain of the offspring that persist into young adulthood. Reactive gliosis correlates positively with hippocampal synaptic decline and cognitive impairments. The elevated pro-inflammatory cytokine expression at the critical period of hippocampal synaptic maturation suggests that neuroinflammation might drive the synaptic and cognitive decline in the offspring of GDM dams. The importance of microglia in this process is supported by the reduced Cx3CR1 expression as an indication of the loss of microglial control of inflammatory responses and phagocytosis and synaptic pruning in GDM offspring.

Keywords: Gestational diabetes mellitus; Hippocampus; Microglia; Neurodevelopmental disorder; Neuroinflammation; Recognition memory; Synapse.

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Figures

Fig. 1
Fig. 1
Gestational diabetes mellitus induces atypically exploratory and inattentive behavior and impairs recognition memory in offspring. Behavioral test analysis of 14- to 15-week-old rats. a, b Open field heat map demonstrates that GDM offspring on postnatal HFS diet stayed more in the center of the field. # p < 0.05, compared to lean LF cohorts, n = 8–10. c–e GDM offspring also showed reduced rearing, but their moving activity was not different from the lean offspring cohorts. f, g In NOR, GDM offspring failed to differentiate the familiar object from the novel object presented to them a day after the training, while lean offspring spent significantly more time exploring the novel object on test day.*p < 0.05, n = 6–10
Fig. 2
Fig. 2
Gestational diabetes mellitus reduces hippocampal synaptic protein expression and deranges CA1 neuronal layer, when combined with postnatal HFS diet. a, b, e Micrographs of 15-week-old rat hippocampus with NeuN and synaptophysin. a–d NeuN staining did not show clear reduction in neuronal numbers, but changes in CA1 pyramidal neuronal layer thickness and density were evident. e, f Hippocampal CA1 area in GDM offspring had decreased synaptophysin expression suggesting decreased synaptic terminal integrity. *p ≤ 0.05 compared to lean, n = 6
Fig. 3
Fig. 3
Gestational diabetes mellitus induces neuroinflammation in the offspring brain (at 20E). Micrographs of immunostaining with Iba1 and GFAP demonstrate elevated neuroinflammation status throughout the rat embryo brain. a, b Iba1 allows visualization of microglial morphology. The microglia of embryos from GDM dams have more activated, amoeboid morphology compared to embryos from dams with healthy pregnancy of which majority possess resting, ramified microglial morphology. d Increase in GFAP expression seen in embryos of GDM dams reflects on astroglial activation and chronic inflammation. Quantitative analysis of hippocampal Iba1-positive cells (c) and GFAP staining (e) confirms the elevated neuroinflammation in GDM offspring. f Cytokine analysis demonstrates increase in pro-inflammatory cytokines (IFNγ, TNFα, IL-1, MCP-1), while only IL-4 was elevated from the anti-inflammatory cytokines (IL-4, IL-10, IL-13). *p ≤ 0.05 compared to lean, n = 6
Fig. 4
Fig. 4
Gestational diabetes mellitus promotes neuroinflammation in offspring (at 15 weeks of age). a Micrographs of Iba1 and GFAP immunostainings from 15-week-old rat hippocampi. b, c Iba1 staining allows visualization of microglial morphology and number of microglia/macrophages present in the hippocampus. Microglia in offspring from lean dams possess resting, ramified morphology, whereas GDM exposure reduced microglial ramification, as evident by increase in hypertrophic morphology and “fully activated” amoeboid morphology in GDM offspring brain. d GDM offspring also showed increased GFAP expression which demonstrates astroglial activation upon GDM. e Cytokine analysis of 15-week-old rat brain cortical section including the hippocampi, cortex, and hypothalamus does not indicate big changes in cytokine profiles between groups. f, g CX3CR1 expression was significantly reduced in GDM-HFS offspring brain. # p ≤ 0.05 compared to lean LF, *p ≤ 0.05 as indicated, n = 6
Fig. 5
Fig. 5
High glucose and palmitic acid exposure stimulates pro-inflammatory response in primary microglial cultures. a–f Responses of cultured microglia exposed to conditions mimicking T2D; utilizing glucose and fatty acid levels find in T2D vs. healthy patients. High fat (HF; 1.2 mM palmitic acid) alone and with elevated glucose (HG; 16.7 mM) induces microglial transformation towards activated amoeboid morphology (a), stimulates microglial proliferation (b), triggers release of nitric oxide (NO) (c), production of superoxide (SO) (d), and alters pro-inflammatory (e) and anti-inflammatory (b) cytokine release profiles in primary microglial cell cultures. # p ≤ 0.05 compared to control (NG), *p ≤ 0.05 as indicated, n = 3-6
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